PROJECT SUMMARY Maladaptive behaviors are a staple of anxiety disorders, yet how specific neural populations drive these behav- iors is unclear. Despite the high prevalence of anxiety disorders and their devastating burden, truly effective therapeutics are still lacking; understanding the underlying pathophysiology is critical. This is not a trivial feat considering the multitude of regions involved and the complexity of associated behaviors. The bed nucleus of the stria terminalis (BNST) is one region highly implicated in governing adaptive and pathological anxiety states, and medial prefrontal cortical (mPFC) neurons are known to regulate motivated behaviors, including those re- lated to anxiety. How mPFC directly modulates BNST activity in anxiety states though is not known. Therefore, the objective of this proposal is to study how mPFC neurons that project to BNST engage in anxiety-related behaviors. Experiments will employ contemporary tools to study the precise neurocircuitry during expression of specific anxiety-related phenotypes. First, in rodent models, anxiety-related behaviors will be identified in the presence of an anxiogenic stimulus using recently developed computer vision and machine learning tools that define behaviors from subsecond modules (Aim 1.1). This is a highly quantitative approach to analyze behavior that will track commonly studied behaviors and, importantly, novel behaviors that have been overlooked in the literature. Second, the role of BNST-projecting mPFC neurons in anxiety-related behaviors will be assessed using inhibitory and excitatory optogenetics in combination with the previously described behavioral analysis (Aim 1.2). This neural population will be targeted using new viral strategies that allow for projection specific targeting. Finally, the physiological neural dynamics of these mPFC neurons during the expression of anxiety- related behaviors will be visualized using mountable, miniature microscopes in mice expressing the genetically encoded calcium indicator GCaMP6s (Aim 2). This activity will be assessed with concurrent behavioral module recording to understand the neuronal dynamics during anxiety-related behaviors. Collectively, these experiments will determine how BNST-projecting mPFC neurons govern expression of the wide gamut of anxiety-related behaviors. Further, the culmination of this study and studies across the neuroscientific community will bear a clearer understanding of how anxiety-related behaviors arise in mental illnesses and will drive development of promising therapeutics.